Glyphosate tolerant wheat plant 33391 and compositions and methods for detection thereof

ABSTRACT

The present invention provides a DNA construct composition that relates to transgenic glyphosate tolerant wheat plants. The invention relates to the wheat plant 33391, the progeny thereof and to methods for the detection of wheat plant 33391 and its progeny and compositions thereof.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/236,653, filed Sep. 29, 2000 and U.S. ProvisionalApplication No. 60/236,762 filed Sep. 29, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of plant molecularbiology, more specifically the invention relates to a DNA construct forconferring improved glyphosate tolerance to a wheat plant. The inventionmore specifically relates to a glyphosate tolerant wheat plant 33391 andprogeny thereof and to assays for detecting the presence of wheat plant33391 DNA in a sample and compositions thereof.

BACKGROUND OF THE INVENTION

[0003] Wheat is an important crop and is a primary food source in manyareas of the world. The methods of biotechnology have been applied towheat for improvement of the agronomic traits and the quality of theproduct. One such agronomic trait is herbicide tolerance, in particular,tolerance to glyphosate herbicide. This trait in wheat is conferred bythe expression of a transgene in the wheat plants (Zhou et al., PlantCell Rep. 15:159-163, 1995). The expression of foreign genes in plantsis known to be influenced by their chromosomal position, perhaps due tochromatin structure (e.g., heterochromatin) or the proximity oftranscriptional regulation elements (e.g., enhancers) close to theintegration site (Weising et al., Ann. Rev. Genet 22:421-477, 1988). Forthis reason, it is often necessary to screen a large number of events inorder to identify an event characterized by optimal expression of aintroduced gene of interest. For example, it has been observed in plantsand in other organisms that there may be a wide variation in levels ofexpression of an introduced gene among events. There may also bedifferences in spatial or temporal patterns of expression, for example,differences in the relative expression of a transgene in various planttissues, that may not correspond to the patterns expected fromtranscriptional regulatory elements present in the introduced geneconstruct. For this reason, it is common to produce hundreds tothousands of different events and screen those events for a single eventthat has desired transgene expression levels and patterns for commercialpurposes. An event that has desired levels or patterns of transgeneexpression is useful for introgressing the transgene into other geneticbackgrounds by sexual outcrossing using conventional breeding methods.Progeny of such crosses maintain the transgene expressioncharacteristics of the original transformant. This strategy is used toensure reliable gene expression in a number of varieties that are welladapted to local growing conditions.

[0004] It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest. In addition, a method for detecting aparticular event would be helpful for complying with regulationsrequiring the premarket approval and labeling of foods derived fromrecombinant crop plants, for example. It is possible to detect thepresence of a transgene by any well known nucleic acid detection methodsuch as the polymerase chain reaction (PCR) or DNA hybridization usingnucleic acid probes. These detection methods generally focus onfrequently used genetic elements, such as promoters, terminators, markergenes, etc. As a result, such methods may not be useful fordiscriminating between different events, particularly those producedusing the same DNA construct unless the sequence of chromosomal DNAadjacent to the inserted DNA (“flanking DNA”) is known. Anevent-specific PCR assay is discussed, for example, by Windels et al.(Med. Fac. Landbouww, Univ. Gent 64/5b:459-462, 1999), who identifiedglyphosate tolerant soybean event 40-3-2 by PCR using a primer setspanning the junction between the insert and flanking DNA, specificallyone primer that included sequence from the insert and a second primerthat included sequence from flanking DNA.

[0005] This invention relates to the improved glyphosate herbicidetolerant wheat (Triticum aestivum) plant 33391 and to a DNA plantexpression construct of wheat plant 33391 and the detection of thetransgene/genomic insertion region in wheat 33391 and progeny thereof.

SUMMARY OF THE INVENTION

[0006] According to one aspect of the invention, a DNA construct isprovided that when expressed in wheat plant cells and wheat plantsconfers improved tolerance to glyphosate herbicide. This inventionrelates to the methods for producing and selecting a glyphosate tolerantwheat plant containing the DNA construct pMON30139. The DNA construct,pMON30139 consists of two transgene expression cassettes. The firstexpression cassette consists of a rice (Oryzae sativa) actin 1 promoter(P-Os.Act1) and intron (I-Os.Act1) operably joined to an ArabidopsisEPSPS chloroplast transit peptide sequence (TS-At.EPSPS), operablyconnected to a gene (AGRTU.aroA:CP4) encoding a glyphosate resistant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) isolated fromAgrobacterium tumefaciens (AGRTU) sp. strain CP4, operably connected toa nopaline synthase transcriptional terminator (T-AGRTU.nos). The secondtransgene expression cassette consists of the cauliflower mosaic virus(CaMV) 35S promoter (P-CaMV.35S:en) containing a tandem duplication ofthe enhancer region, operably connected to a Zea mays Hsp70 intron(I-Zm.Hsp70), operably connected to a nucleic acid sequence encoding anArabidopsis thaliana EPSPS chloroplast transit peptide sequence,operably connected to a gene encoding a glyphosate resistant5-enol-pyruvylshikimate-3-phosphate synthase isolated from Agrobacteriumtumefaciens sp. strain CP4, operably connected to a nopaline synthasetranscriptional terminator. These expression cassettes are in tandem andflanked by DNA regions that contain Agrobacterium tumefaciens DNAsequences (RB and LB) as a components of the process that is used in anAgrobacterium mediated method to insert of the expression cassettes intoa wheat genome.

[0007] According to another aspect of the invention, wheat 33391 seedcomprising such DNA molecules are provided as deposited with the ATCC,accession #PTA-2347. This aspect of the invention thus relates to theseeds of wheat 33391, to the plants of wheat 33391, to the plant partsof wheat 33391 that includes pollen and ovules, and to the methods forproducing an improved glyphosate tolerant wheat plant by crossing thewheat plant 33391 with itself or another wheat plant.

[0008] According to another aspect of the invention, compositions andmethods are provided for detecting the presence of the transgene/genomicinsertion region from wheat 33391 plants and seeds. According to oneaspect of the invention, DNA molecules are provided that comprise atleast one transgene/genomic insertion region sequence of wheat 33391selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6 andcomplements thereof, wherein an insertion region sequence spans thejunction between heterologous DNA inserted into the wheat genome and DNAfrom the wheat genome flanking the insertion site and is diagnostic forthe event. Included are DNA sequences that comprise a sufficient lengthof polynucleotides of transgene insert sequence and a sufficient lengthof polynucleotides of wheat genomic sequence from wheat 33391 of SEQ IDNO:5 that are useful as primer sequences for the production of anamplicon product diagnostic for wheat 33391. Included are DNA sequencesthat comprise a sufficient length of polynucleotides of transgene insertsequence and a sufficient length of polynucleotides of wheat genomicsequence from wheat 33391 of SEQ ID NO:6 that are useful as primersequences for the production of an amplicon product diagnostic for wheat33391.

[0009] According to another aspect of the invention DNA molecules areprovided that are diagnostic for wheat 33391. This aspect of theinvention is directed to the wheat 33391 containing at least one novelDNA molecule. DNA molecules comprising nucleic acid primers are providedthat provide at least one novel DNA amplicon product of wheat 33391consisting of SEQ ID NO:7 and SEQ ID NO:8, or the complements thereof.Such DNA amplicons are diagnostic for wheat 33391. Nucleic acidamplification of genomic DNA of the wheat 33391 produces an ampliconcomprising such diagnostic DNA sequences. The invention providesisolated DNA molecules that comprise a sufficient length of transgeneinsert sequence and a sufficient length of wheat genomic sequence fromwheat 33391 to function as primer sequences for the production of anamplicon product diagnostic for wheat 33391.

[0010] According to another aspect of the invention, methods ofdetecting the presence of DNA corresponding to the wheat 33391 in asample are provided. Such methods comprise: (a) contacting the samplecomprising DNA with a primer set that, when used in a nucleic-acidamplification reaction with genomic DNA from wheat 33391, produces anamplicon that is diagnostic for wheat 33391; (b) performing a nucleicacid amplification reaction, thereby producing the amplicon; and (c)detecting the amplicon.

[0011] According to another aspect of the invention, a kit is providedfor the detection of wheat 33391. The kit includes at least one DNAsequence of sufficient length of polynucleotides complementary to SEQ IDNO:5 or SEQ ID NO:6, wherein the DNA sequences are useful as primers orprobes that hybridize to isolated DNA from wheat 33391 or its progeny.

[0012] According to another aspect of the invention, methods ofproducing a wheat plant with improved tolerance to glyphosate areprovided that comprise the steps of: (a) sexually crossing a firstparental wheat line comprising the pMON30139 construct that confersimproved tolerance to application of glyphosate, and a second parentalwheat line that lacks glyphosate tolerance, thereby producing aplurality of progeny plants; and (b) selecting a progeny plant thattolerates application of glyphosate. Such methods are useful forintrogressing the glyphosate tolerant trait into different geneticbackgrounds. Such methods may optionally comprise the further step ofback-crossing the progeny plant to the second parental wheat line toproduce a wheat plant that tolerates application of glyphosate.

[0013] The foregoing and other aspects of the invention will become moreapparent from the following detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1. Plasmid map of pMON30167

[0015]FIG. 2. Plasmid map of pMON42411

[0016]FIG. 3. Plasmid map of pMON30139

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] This application claims the benefit of U.S. ProvisionalApplication No. 60/236,653, filed Sep. 29, 2000 and U.S. ProvisionalApplication No. 60/236,762 filed Sep. 29, 2000. The followingdefinitions and methods are provided to better define the presentinvention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin,Genes V, Oxford University Press: New York, 1994. The nomenclature forDNA bases as set forth at 37 CFR § 1.822 is used.

[0018] As used herein, the term “wheat” means Triticum aestivum(including spring, winter, and facultative wheat varieties) any otherwheat species that can be bred with Triticum aestivum, including but notlimited to durum wheat (Triticum durum), spelt (Triticum spelta), andemmer (Triticum dicoccum). Also encompassed are plants that are producedby conventional techniques using Triticum aestivum as a parent in asexual cross with a non-Triticum species (such as rye [Secale cereale]),including but not limited to triticale.

[0019] As used herein, the term “comprising” means “including but notlimited to”.

[0020] “Glyphosate” refers to N-phosphonomethylglycine and its salts.Glyphosate is the active ingredient of Roundup® herbicide (Monsanto Co,St. Louis, Mo.). Treatments with “glyphosate herbicide” refer totreatments with the Roundup®, Roundup Ultra® herbicide or any otherformulation containing glyphosate. For the purposes of the presentinvention, the term “glyphosate” includes any herbicidally active formof N-phosphonomethylglycine (including any salt thereof) and other formsthat result in the production of the glyphosate anion in plants.Treatments with “glyphosate” refer to treatments with the Roundup® orRoundup Ultra® herbicide formulation, unless otherwise stated. Planttransformation and regeneration in tissue culture use glyphosate orsalts of glyphosate. Whole plant assays use formulated Roundup® orRoundup Ultra®. Additional formulations with herbicide activity thatcontain N-phosphonomethylglycine or any of its salts are herein includedas a glyphosate herbicide.

[0021] A transgenic “event” is produced by transformation of plant cellswith heterologous DNA, i.e., a nucleic acid construct that includes atransgene of interest, regeneration of a population of plants resultingfrom the insertion of the transgene into the genome of the plant, andselection of a particular plant characterized by insertion into aparticular genome location. The term “event” refers to the originaltransformant and progeny of the transformant that include theheterologous DNA. The term “event” also refers to progeny produced by asexual outcross between the transformant and another variety thatinclude the heterologous DNA. Even after repeated back-crossing to arecurrent parent, the inserted DNA and flanking DNA from the transformedparent is present in the progeny of the cross at the same chromosomallocation. The term “event” also refers to DNA from the originaltransformant and progeny thereof comprising the inserted DNA andflanking genomic sequence immediately adjacent to the inserted DNA thatwould be expected to be transferred to a progeny that receives insertedDNA including the transgene of interest as the result of a sexual crossof one parental line that includes the inserted DNA (e.g., the originaltransformant and progeny resulting from selfing) and a parental linethat does not contain the inserted DNA. The “event” of the presentinvention comprises wheat 33391 seed having ATCC accession No. PTA-2347and wheat plants grown from the wheat 33391 and progeny thereof. A wheatplant that tolerates a sufficient amount of glyphosate herbicide tocontrol the weeds in a field without affecting the wheat plant can bebred by first sexually crossing a first parental wheat plant consistingof a wheat plant containing the expression cassettes of pMON30139 thatconfers improved tolerance to application of glyphosate herbicide, and asecond parental wheat plant that lacks the tolerance to glyphosateherbicide, thereby producing a plurality of first progeny plants; andthen selecting a first progeny plant that is tolerant to application ofglyphosate herbicide; and selfing the first progeny plant, therebyproducing a plurality of second progeny plants; and then selecting fromthe second progeny plants a glyphosate herbicide tolerant plant. Thesesteps can further include the back-crossing of the first glyphosatetolerant progeny plant or the second glyphosate tolerant progeny plantto the second parental wheat plant or a third parental wheat plant,thereby producing a wheat plant that tolerates the application ofglyphosate herbicide. A wheat crop comprising wheat 33391 seeds orprogeny thereof can be planted in a field and treated with a sufficientamount of glyphosate herbicide to control the weeds withoutsignificantly affecting the wheat crop. A sufficient amount ofglyphosate herbicide is about 8 ounces/acre or more, 16 ounces/acre ormore, 32 ounces/acre or more, or 64 ounces/acre or more. Any glyphosatecontaining herbicide formulation can be used to control weeds in a wheatcrop comprising wheat 33391 plants or progeny thereof.

[0022] It is also to be understood that two different transgenic plantscan also be mated to produce offspring that contain two independentlysegregating added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes thatencode a polypeptide of interest. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated, as isvegetative propagation. Descriptions of other breeding methods that arecommonly used for different traits and crops can be found in one ofseveral references, e.g., Fehr, in Breeding Methods for CultivarDevelopment, Wilcox J. ed., American Society of Agronomy, Madison Wis.(1987) herein incorporated by reference in its entirety; Poehlman, J. M.(1987); Breeding Field Crops, 3rd ed. Van Nostrand Reinhold, N.Y.,Knott, D. R. (1987); herein incorporated by reference in its entiretyThe Application of Breeding Procedures to Wheat, p. 419-427. In E. G.Heyne (ed.) In “Wheat and Wheat Improvement”, Madison, Wis. hereinincorporated by reference in its entirety. Backcross breeding has beenused to transfer genes for a simply inherited, highly heritable traitinto a desirable homozygous cultivar or inbred line, which is therecurrent parent. The source of the trait to be transferred is calledthe donor parent. The resulting plant is expected to have the attributesof the recurrent parent (e.g., cultivar) and the desirable traittransferred from the donor parent. After the initial cross, individualspossessing the phenotype of the donor parent are selected and repeatedlycrossed (backcrossed) to the recurrent parent. The resulting parent isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent.

[0023] The DNA molecules of the present invention can by used asmolecular markers in a marker assisted breeding (MAB) method. DNAmolecules of the present invention can be used in methods, such as, AFLPmarkers, RFLP markers, RAPD markers, SNPs, and SSRs that identifygenetically linked agronomically useful traits as described by Walton,Seed World 22-29 (July, 1993), the entirety of which is hereinincorporated by reference; Burow and Blake, Molecular Dissection ofComplex Traits, 13-29, Eds. Paterson, CRC Press, New York (1988), theentirety of which is herein incorporated by reference). The improvedglyphosate tolerance trait of wheat plant 33391 can be tracked in theprogeny of a cross with wheat plant 33391 and any other wheat cultivaror variety using the MAB methods. The DNA molecules are markers for thistrait and in MAB methods that are well known in the art can be used totrack glyphosate tolerance in wheat where wheat plant 33391 was a parentor ancestor.

[0024] A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, e.g., a radioactiveisotope, ligand, chemiluminescent agent, or enzyme. Such a probe iscomplementary to a strand of a target nucleic acid, in the case of thepresent invention, to a strand of genomic DNA from wheat event 33391(whether from a wheat plant or from a sample that includes DNA from theevent). Probes according to the present invention include not onlydeoxyribonucleic or ribonucleic acids but also polyamides and otherprobe materials that bind specifically to a target DNA sequence and canbe used to detect the presence of that target DNA sequence.

[0025] “Primers” are isolated nucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, then extended alongthe target DNA strand by a polymerase, e.g., a DNA polymerase. Primerpairs or sets can be used for amplification of a nucleic acid sequence,e.g., by the polymerase chain reaction (PCR) or other conventionalnucleic-acid amplification methods.

[0026] Probes and primers are generally 8 polynucleotides or more inlength, 18 polynucleotides or more, 24 polynucleotides or more, 30polynucleotides or more. Polynucleotides useful as probes and primersthat are of sufficient length to hybridize specifically to a targetsequence under stringent conditions for hybridization. Probes andprimers according to the present invention have complete sequencesimilarity with the target sequence, although probes differing from thetarget sequence and that retain the ability to hybridize to targetsequences may be designed by conventional methods.

[0027] Methods for preparing and using probes and primers are described,for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”) hereinincorporated by reference in its entirety; Current Protocols inMolecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates) (hereinafter,“Ausubel et al., 1992) herein incorporated by reference in its entirety;and Innis et al., PCR Protocols: A Guide to Methods and Applications,Academic Press: San Diego, 1990 herein incorporated by reference in itsentirety. PCR-primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5, ® 1991, Whitehead Institute for BiomedicalResearch, Cambridge, Mass.) herein incorporated by reference in itsentirety.

[0028] Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm (and, if necessary, to correct)the disclosed sequences by conventional methods, e.g., by re-cloning andsequencing such sequences.

[0029] The nucleic-acid probes and primers of the present inventionhybridize under stringent conditions to a target DNA sequence. Anyconventional nucleic acid hybridization or amplification method can beused to identify the presence of DNA from a transgenic event in asample.

[0030] The term “stringent conditions” is functionally defined withregard to the hybridization of a nucleic-acid probe to a target nucleicacid (i.e., to a particular nucleic-acid sequence of interest) by thespecific hybridization procedure discussed in Sambrook et al., 1989, at9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52, 9.56-9.58herein incorporated by reference in its entirety; Kanehisa, (Nucl. AcidsRes. 12:203-213, 1984, herein incorporated by reference in itsentirety); and Wetmur and Davidson, (J. Mol. Biol. 31:349-370, 1988,herein incorporated by reference in its entirety). Accordingly, thenucleotide sequences of the invention may be used for their ability toselectively form duplex molecules with complementary stretches of DNAfragments. Depending on the application envisioned, one will desire toemploy varying conditions of hybridization to achieve varying degrees ofselectivity of probe towards target sequence. For applications requiringhigh selectivity, one will typically desire to employ relativelystringent conditions to form the hybrids, e.g., one will selectrelatively low salt and/or high temperature conditions, such as providedby about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. toabout 70° C. A stringent conditions, for example, is to wash thehybridization filter at least twice with high-stringency wash buffer(0.2×SSC, 0.1% SDS, 65° C.). Appropriate stringency conditions whichpromote DNA hybridization, for example, 6.0×sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C.,are known to those skilled in the art or can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged. Such selective conditions tolerate little, if any, mismatchbetween the probe and the template or target strand. Detection of DNAsequences via hybridization is well-known to those of skill in the art,and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 areexemplary of the methods of hybridization analyses.

[0031] Regarding the amplification of a target nucleic-acid sequence(e.g., by PCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic-acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon.

[0032] The term “specific for (a target sequence)” indicates that aprobe or primer hybridizes under stringent hybridization conditions onlyto the target sequence in a sample comprising the target sequence.

[0033] As used herein, “amplified DNA” or “amplicon” refers to theproduct of nucleic acid amplification of a target nucleic acid sequencethat is part of a nucleic acid template. For example, to determinewhether the wheat plant resulting from a sexual cross contains antransgenic event, genomic DNA from a wheat plant may be subjected tonucleic acid amplification using a primer pair that includes a primerderived from flanking sequence in the genome of the plant adjacent tothe insertion site of inserted heterologous DNA and a second primerderived from the inserted heterologous DNA to produce an amplicon thatis diagnostic for the presence of the event. The amplicon is of a lengthand has a sequence that is diagnostic for the event. Alternatively, aprimer pair can be derived from flanking sequence on both sides of theinserted DNA so as to produce an amplicon that includes the entireinsert.

[0034] Nucleic acid amplification can be accomplished by any of thevarious nucleic acid amplification methods known in the art, includingthe polymerase chain reaction (PCR). A variety of amplification methodsare known in the art and are described, inter alia, in U.S. Pat. Nos.4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods andApplications, ed. Innis et al., Academic Press, San Diego, 1990. Anywell known method for nucleic acid amplification may be used in thepractice of the present invention. The sequence of the heterologous DNAinsert or flanking sequence from wheat 33391 event, ATCC accession No.PTA-2347 can be verified (and corrected if necessary) by amplifying suchsequences from the event using primers derived from the sequencesprovided herein followed by standard methods of DNA sequencing of thePCR amplicon or of the cloned DNA molecule.

[0035] The amplicon produced by these methods may be detected by aplurality of techniques. Agarose gel electrophoresis and staining withethidium bromide is a common well known method of detecting DNAamplicons. Another method is Genetic Bit Analysis (Nikiforov, et al.Nucleic Acid Res. 22:4167-4175, 1994) where an DNA oligonucleotide isdesigned which overlaps both the adjacent flanking genomic DNA sequenceand the inserted DNA sequence. The oligonucleotide is immobilized inwells of a microtiter plate. Following PCR of the region of interest(using one primer in the inserted sequence and one in the adjacentflanking genomic sequence), a single-stranded PCR product can behybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labelledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

[0036] An additional method is the Pyrosequencing technique as describedby Winge (Innov. Pharma. Tech. 00:18-24, 2000). In this method anoligonucleotide is designed that overlaps the adjacent genomic DNA andinsert DNA junction. The oligonucleotide is hybridized tosingle-stranded PCR product from the region of interest (one primer inthe inserted sequence and one in the flanking genomic sequence) andincubated in the presence of a DNA polymerase, ATP, sulfurylase,luciferase, apyrase, adenosine 5′ phosphosulfate and luciferin. DNTPsare added individually and the incorporation results in a light signalwhich is measured. A light signal indicates the presence of thetransgene/flanking sequence due to successful amplification,hybridization, and single or multi-base extension.

[0037] Fluorescence Polarization as described by Chen, et al., (GenomeRes. 9:492-498, 1999) is a method that can be used to detect theamplicon of the present invention. Using this method an oligonucleotideis designed which overlaps the genomic flanking and inserted DNAjunction. The oligonucleotide is hybridized to single-stranded PCRproduct from the region of interest (one primer in the inserted DNA andone in the flanking genomic DNA sequence) and incubated in the presenceof a DNA polymerase and a fluorescent-labeled ddNTP. Single baseextension results in incorporation of the ddNTP. Incorporation can bemeasured as a change in polarization using a fluorometer. A change inpolarization indicates the prescence of the transgene/flanking sequencedue to successful amplification, hybridization, and single baseextension.

[0038] Taqman® (PE Applied Biosystems, Foster City, Calif.) is describedas a method of detecting and quantifying the presence of a DNA sequenceand is fully understood in the instructions provided by themanufacturer. Briefly, a FRET oligonucleotide probe is designed whichoverlaps the genomic flanking and insert DNA junction. The FRET probeand PCR primers (one primer in the insert DNA sequence and one in theflanking genomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Hybridization of the FRET probe results incleavage and release of the fluorescent moiety away from the quenchingmoiety on the FRET probe. A fluorescent signal indicates the presence ofthe flanking/transgene sequence due to successful amplification andhybridization.

[0039] Molecular Beacons have been described for use in sequencedetection as in Tyangi, et al. (Nature Biotech. 14:303-308, 1996)Briefly, a FRET oligonucleotide probe is designed that overlaps theflanking genomic and insert DNA junction. The unique structure of theFRET probe results in it containing secondary structure that keeps thefluorescent and quenching moieties in close proximity. The FRET probeand PCR primers (one primer in the insert DNA sequence and one in theflanking genomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal results. Afluorescent signal indicates the presence of the flanking/transgenesequence due to successful amplification and hybridization.

[0040] The following examples are included to demonstrate examples ofcertain preferred embodiments of the invention. It should be appreciatedby those of skill in the art that the techniques disclosed in theexamples that follow represent approaches the inventors have foundfunction well in the practice of the invention, and thus can beconsidered to constitute examples of preferred modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments that are disclosed and still obtain a like or similar resultwithout departing from the spirit and scope of the invention.

EXAMPLE 1

[0041] The transgenic wheat plants are generated byAgrobacterium-mediated transformation of wheat embryos by the method ofCheng et al. (Plant Physiol. 115:971-980, 1997) using the binary vectorsof the present invention and a modification of the glyphosate selectionconditions of Zhou et al. (Plant Cell Rep. 15:159-163, 1995). Othermethods of wheat transformation are known to those skilled in the art ofwheat transformation, such as, gene gun or particle bombardment and canbe used to insert the expression cassettes of the present invention intothe genome of wheat cells. The T-DNA of pMON30139 (FIG. 3) contains twoexpression cassettes that collectively confer a high level of toleranceto glyphosate herbicide. The first transgene expression cassettecomprises DNA sequences of the rice actin 1 promoter and intron(P-Os.Act1 and I-Os.Act1, U.S. Pat. No. 5,641,876, herein incorporatedby reference in its entirety), operably connected to a DNA sequenceencoding an Arabidopsis thaliana EPSPS chloroplast transit peptide(TS-At.EPSPS:CTP2, Klee et al., Mol. Gen. Genet. 210:47-442, 1987,herein incorporated by reference in its entirety), operably connected toa DNA sequence encoding a glyphosate resistant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) isolated fromAgrobacterium tumefaciens sp. strain CP4 (AGRTU.aroA gene, U.S. Pat. No.5,633,435, herein incorporated by reference in its entirety), operablyconnected to a DNA sequence of a nopaline synthase transcriptionalterminator (T-AGRTU.nos, Fraley et al., Proc. Natl. Acad. Sci. USA80:4803-4807,1983, herein incorporated by reference in its entirety).The second transgene expression cassette comprises a DNA sequence of acauliflower mosaic virus 35S promoter containing a tandem duplication ofthe enhancer region (P-CaMV.35S:en, Kay et al., Science 236:1299-1302,1987; U.S. Pat. No. 5,164,316, herein incorporated by reference in itsentirety), operably connected to a DNA sequence of a Zea mays Hsp70intron (I-Zm.Hsp70, U.S. Pat. No. 5,424,412, herein incorporated byreference in its entirety), operably connected to a DNA sequenceencoding an Arabidopsis thaliana EPSPS chloroplast transit peptidesequence (TS-At.EPSPS, Klee et al., Mol. Gen. Genet. 210:47-442, 1987),operably connected to a DNA sequence encoding a glyphosate resistant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) isolated fromAgrobacterium tumefaciens sp. strain CP4 (AGRTU.aroA:CP4 gene, U.S. Pat.No. 5,633,435), operably connected to a DNA sequence of a nopalinesynthase transcriptional terminator (T-AGRTU.nos, Fraley et al., Proc.Natl. Acad. Sci. USA 80:4803-4807, 1983).

[0042] pMON30167 (FIG. 1) is a single expression cassette identical tothe first transgene expression cassette of pMON30139 as described above.pMON42411 (FIG. 2) is a single expression cassette identical to thesecond expression cassette of pMON30139 as described above.

[0043] After incubation of wheat cells with the Agrobacterium cellscontaining pMON42411, pMON30167 and pMON30139 constructs,glyphosate-tolerant transgenic wheat calli were selected on mediacontaining 2 mM glyphosate for 1 week followed by transfer to adifferentiation media with 0.1 mM glyphosate for 2 weeks and finallytransfer to regeneration media with 0.02 mM glyphosate +0.1 μM aromaticamino acids.

[0044] Two hundred eight-four wheat events were produced fromtransformation with pMON42411, pMON30167 and pMON30139. These plantsfrom pMON30139 and pMON30167 were sprayed once with 64 ounces/acre rateof glyphosate herbicide (Roundup Ultra™)/acre) to select lines forvegetative and reproductive tolerance to glyphosate herbicide (Table 1).Plants from pMON42411 were sprayed twice with 64 ounces/acre rate ofglyphosate herbicide. Selection of transformed wheat plants with thesingle expression cassettes of pMON42411 and pMON30167 resulted in a lowpercentage (1.4% and 3.2%, respectively) of wheat plants with bothvegetative and reproductive tolerance. Only 3/134 plants from theseconstructs had acceptable levels of glyphosate herbicide tolerance. Incontrast, transformed wheat plants containing the double expressioncassette of pMON30139 produced a high percentage (16%) of plants withboth vegetative and reproductive tolerance (24/150).

[0045] Wheat event 33391(hence forth referred to as wheat plant 33391 orwheat 33391 and includes all plant parts and seed of this plant) wasselected from the 150 transgenic wheat events produced fromtransformation with pMON30139. Twenty-four events were selected fromthis population that demonstrated improved vegetative and reproductiveglyphosate tolerance. Further evaluation of these 24 events wasconducted for agronomic performance and the presence of a single intactinsertion. Wheat 33391 was selected from this population of events. Thegreenhouse and field evaluations of wheat 33391 and progeny derived fromwheat 33391 indicated that this transgenic insertion confers glyphosatetolerance that exceeds commercial specifications of full vegetative andreproductive tolerance to 340 g glyphosate/acre (840 gglyphosate/hectare; 32 oz of Roundup Ultra/acre) with two-fold safetymargin when applied at the 3-5 leaf stage. TABLE 1 Comparison ofefficacy of single and double expression cassettes for conferringglyphosate tolerance in wheat. # events # events with # veg. tolerantevents with pMON# tested vegetative tolerance reproductive tolerance42411 71 26 1 (1.4%) 30167 63 4 2 (3.5%) 30139 150 104 24 (16%)  

EXAMPLE 2

[0046] Isolation of the corresponding wheat genomic flanking sequence ispossible by a variety of methods known to those skilled in the art (forexample, using the ligated adapters and nested PCR as described in theGenome Walker™ kit, (CloneTech Laboratories, Inc, Palo Alto, Calif.).Genomic DNA from the wheat 33391 was isolated by CTAB purificationmethod (Rogers et al., Plant Mol Biol 5:69-76, 1985). Reagents areavailable commercially (see, for example Sigma Chemical Co., St. Louis,Mo.). The genomic DNA libraries for amplification were preparedaccording to manufacturer instructions (Genome Walker™, CloneTechLaboratories, Inc, Palo Alto, Calif.). In separate reactions, genomicDNA was subjected to restriction enzyme digestion overnight at 37° C.with the following blunt-end endonucleases: Dral, EcoRV, PvuII, ScaI,and StuI (CloneTech Laboratories, Inc. Palo Alto, Calif.). The reactionmixtures were extracted with phenol:chloroform, the DNA was precipitatedby the addition of ethanol to the aqueous phase, pelleted bycentrifugation, then resuspended in Tris-EDTA buffer (10 mM Tris-.HCl,pH 8.0, 1 mM EDTA). The purified blunt-ended genomic DNA fragments werethen ligated to the Genome Walker™ adapters according to themanufacturer's protocol. After ligation of the adapters to the genomicDNA fragments, each reaction was heat treated (70° C. for 5 minutes) toterminate the reaction and then diluted 10-fold in Tris-EDTA buffer. Oneμl of each respective ligation was then amplified in a 50 μl reactionaccording to manufacturer's recommended protocol using anadapter-specific oligonucleotide (supplied by manufacturer) and a wheat33391 transgene-specific oligonucleotide, such as SEQ ID NO: 1, whichanneals near the 5′ end of the P-Os.Act1. The PCR mixture contained 1 μlof respective adapter-ligated library, 1 μl of 10 μM Genome Walker™adapter primer AP1 supplied by manufacturer (5′GTATATCGACTCACTATAGGGC3′,SEQ ID NO:11), 1 μl of 10 μM wheat 33391 transgene specificoligonucleotide (SEQ ID NO:1), 1 μl of 10 mM deoxyribonucleotides, 5 μlof 10×PCR buffer containing MgCl₂, 0.5 μl (2.5 units) of Taq DNApolymerase (Boehringer Mannheim Biochemicals, Indianapolis, Ind.), andH₂O to 50 μl. The PCR reactions were performed in a thermocycler usingcalculated temperature control and the following cycling conditions: 1cycle of 94° C. for 1 minutes; 7 cycles of (94° C. for 2 seconds, 70° C.for 3 minutes); 37 cycles of (94° C. for 2 seconds, 65° C. for 3minutes); 1 cycle of 65° C. for 10 minutes. One μl of each primaryreaction was then amplified in a secondary amplification using a“nested” adapter-specific oligonucleotide (supplied by manufacturer) anda “nested” transgene-specific oligonucleotide such as SEQ ID NO:2, whichanneals to P-Os.Act1 upstream of the primer used in the primaryreaction. The PCR mixture for secondary PCR contained 1 μl of respectiveprimary PCR products, 1 μl of 10 μM Genome Walker™ nested adapter primerAP2 supplied by manufacturer (5′ACTATAGGGCACGCGTGGT3′, SEQ ID NO:12), 1μl of 10 μM wheat 33391 transgene-specific nested oligonucleotide (SEQID NO:2), 1 μl 10 mM deoxyribonucleotides, 5 μl of 10×PCR buffercontaining MgCl₂, 0.5 μl (2.5 units) of Taq DNA polymerase (BoehringerMannheim Biochemicals, Indianapolis, Ind.), and H₂O to 50 μl. The PCRreactions were again performed in a thermocycler using calculatedtemperature control and the following cycling conditions: 1 cycle of 94°C. for 1 minute; 7 cycles of (94° C. for 2 seconds), 70° C. for 3minute; 31 cycles of (94° C. for 2 seconds, 65° C. for 3 minute); 1cycle of 65° C. for 10 minute. PCR products, representing 5′ regionsthat span the junction between the wheat 33391 transgenic insertion andthe neighboring flanking genomic sequence were then purified by agarosegel electrophoresis followed by isolation from the agarose matrix usingthe QIAquick Gel Extraction Kit (catalog #28704, Qiagen Inc., Valencia,Calif.) and then directly cloned into the pGEM-T Easy vector (catalog#A1360, Promega, Madison, Wis.). The identity of the cloned PCR productswas confirmed by DNA sequence analysis (ABI Prism™ 377, PE Biosystems,Foster City, Calif. and DNASTAR sequence analysis software, DNASTARInc., Madison, Wis.).

[0047] Similarly, the wheat 33391 3′ flanking genomic DNA sequence wasamplified and cloned using nested gene specific primers, such as SEQ IDNO:3, and SEQ ID NO:4, that anneal to the T-nos transcriptionalterminator. Two T-nos transcriptional terminators are present in thewheat 33391 transgenic/genomic insertion, one internal in the constructand one at the 3′ end of the construct adjacent to wheat genomicsequence. The PCR products produced in this reaction were sequenced andthe DNA sequence that spans the junction between transgene and flankinggenomic was distinguished from products of the internal T-nos bycomparison to the known genetic element sequences of the pMON30139construct.

[0048] Wheat genomic sequence flanking both sides of the transgeneinsertion site in the wheat genomic was determined for wheat 33391 bysequencing the Genome Walker™-derived amplification products andalignment to known transgene sequence. The sequence of a 399 base pairs(bp) segment around the insertion site was determined at the 5′ end ofthe transgene insertion site. This segment consisted of 257 (bp) ofwheat genomic sequence (nucleotide bases 1-257 of SEQ ID NO:5) and 93 bpof vector backbone sequence (nucleotide bases 258-350 of SEQ ID NO:5)and 49 bp of the 5′ end of the rice Act1 promoter (nucleotide bases251-399 of SEQ ID NO:5). Similarly, DNA sequence was determined for a431 bp segment flanking the 3′ insertion junction (SEQ ID NO:6),beginning with 32 bp of the T-nos transcriptional terminator sequence(nucleotide bases 1-32 of SEQ ID NO:6), 68 bp of vector backbonesequence (nucleotide bases 33-100) and ending with 331 bp of wheatgenomic sequence flanking the transgene insertion site (nucleotide bases101-431 of SEQ ID NO:6). Identification of wheat 33391 was performed byPCR amplification of the transgene/genomic insertion region using oneprimer from transgene sequence and another primer from the wheat genomicflanking sequence. The 5′ transgene/genomic insertion region wasconfirmed by PCR amplification of a DNA amplicon to be unique to wheat33391. This identification was demonstrated by a PCR amplicon generatedby primer 5 (SEQ ID NO:7) and primer 6 (SEQ ID NO:8). Additional primersequences can be synthesized using the DNA sequence shown in SEQ ID NO:5that will generate amplicons of DNA length different than the amplicongenerated by primer 5 and primer 6, but are still diagnostic for wheat33391 and progeny thereof. It is within the ordinary skill in the art ofa plant molecular biologist to select DNA primer sequences from SEQ IDNO:5 and develop stringent conditions for the production of an amplicon.Likewise, those skilled in the art can select DNA primer sequences fromSEQ ID NO:6 that will generate amplicons diagnostic for wheat 33391. Itis within the scope of this invention that DNA primer sequences derivedfrom SEQ ID NO:5 and SEQ ID NO:6 are useful for the isolation ofadditional genomic DNA molecules from wheat 33391 plants, seeds andplant part by the methods disclosed herein or methods known in the artof plant molecular biologist. These additional wheat genomic DNAmolecules can be isolated in a method that uses any portion ofsufficient length of the DNA sequence disclosed in SEQ ID NO:5 and SEQID NO:6 useful as a primer or probe. The additional wheat genomic DNAmolecules can be used as molecular markers diagnostic for wheat 33391.

[0049] DNA sequences that span the junction region of the wheat 33391genomic DNA and the insert DNA of pMON30139 contained within SEQ ID NO:5and SEQ ID NO:6 can be used as probes in a hybridization reaction toidentify DNA derived from wheat 33391. For example, a DNA moleculeuseful as a probe from SEQ ID NO:5 would comprise the nucleotidesequence occurring from position 245-270 or its complement; a DNAmolecule useful as a probe from SEQ ID NO:6 would comprise thenucleotide sequence occurring from position 87-113 or its complement.Those skilled in the art can select nucleotide sequences shorter orlonger in length than those afore described that span the junctionregion and are useful as specific DNA probes or primers for wheat 33391under high stringency conditions.

[0050] The PCR reaction conditions (Table 2) and quality of theextracted wheat 33391 genomic DNA are confirmed by the production of anamplicon by primer 7 (SEQ ID NO:9) and primer 8 (SEQ ID NO:10) andrepresenting an approximately 400 bp DNA fragment from the wheat acetylCoA carboxylase gene (Acc), a single copy endogenous gene within thewheat genome. The controls for this analysis should include a positivecontrol from wheat 33391, a negative control from a wheat plant that isnot wheat 33391, and a negative control that contains no template wheatDNA as shown in Table 2. The assay for the wheat 33391 amplicon can beperformed by using a Stratagene Robocycler, MJ Engine, Perkin-Elmer9700, or Eppendorf Mastercycler Gradient thermocycler as shown in Table3, or by methods and apparatus known to those skilled in the art. TABLE2 PCR procedure and reaction mixture for the confirmation of wheat 333915′ transgene/genomic junction region. Step Reagent Amount Comments 1Nuclease-free water add to final volume of 20 μl — 2 10 × reactionbuffer 2.0 μl 1 × final (with MgCl₂) concentration of buffer, 1.5 mMfinal concentration of MgCl₂ 3 10 mM solution of dATP, 0.4 μl  200 μMfinal dCTP, dGTP, and dTTP concentration of each dNTP 4 Primer 5 (SEQ IDNO: 7) 0.4 μl  0.2 μM final (resuspended in 1 × TE concentration bufferor nuclease-free water to a concentration of 10 μM) 5 Primer 6 (SEQ IDNO: 8) 0.4 μl  0.2 μM final (resuspended in 1 × TE buffer orconcentration nuclease-free water to a concentration of 10 μM) 6 Primer7 (SEQ ID NO: 9) 0.2 μl  0.1 μM final (resuspended in 1 × TE buffer orconcentration nuclease-free water to a concentration of 10 μM) 7 Primer8 (SEQ ID NO: 10) 0.2 μl  0.1 μM final (resuspended in 1 × TE buffer orconcentration nuclease-free water to a concentration of 10 μM) 8 RNase,DNase free (500 ng/μl) 0.1 μl 50 ng/reaction 9 REDTaq DNA polymerase 1.0μl (recommended to switch 1 unit/reaction (1 unit/μl) pipets prior tonext step) 10 Extracted DNA (template): Samples to be analyzedindividual leaves 10-200 ng of genomic DNA pooled leaves (maximum of 200ng of genomic DNA 50 leaves/pool) Negative control  50 ng of wheatgenomic DNA (not wheat 33391) Negative control no template DNA Positivecontrol  50 ng of 33391 genomic DNA

[0051] TABLE 3 Suggested PCR parameters for different thermocyclersGently mix and, if needed (no hot top on thermocycler), add 1-2 drops ofmineral oil on top of each reaction. Proceed with the PCR in aStratagene Robocycler, MJ Engine, Perkin-Elmer 9700, or EppendorfMastercycler Gradient thermocycler using the following cyclingparameters. Cycle No. Settings: Stratagene Robocycler  1 94° C. 3minutes 38 94° C. 1 minute 63° C. 1 minute 72° C. 1 minute and 30seconds  1 72° C. 10 minutes Cycle No. Settings: MJ Engine orPerkin-Elmer 9700  1 94° C. 3 minutes 38 94° C. 10 seconds 63° C. 30seconds 72° C. 1 minute  1 72° C. 10 minutes Cycle No. Settings:Eppendorf Mastercycler Gradient  1 94° C. 3 minutes 38 94° C. 15 seconds63° C. 15 seconds 72° C. 1 minute  1 72° C. 10 minutes

EXAMPLE 3

[0052] The expression of the glyphosate resistant EPSPS protein (CP4EPSPS) from aroA:CP4 gene can be detected by immunological methods(Rogan et al., Food Control 10:407-414, 1999, herein incorporated byreference in its entirety) from plant tissue extracts. lmmunologicalmethods such as western blots, strip tests, and enzyme linkedimmunosorbent assays (ELISA) have been developed to specifically detectthe protein expressed from the aroA:CP4 gene contained in plantexpression vectors transformed into plants. Reagents that include thepolyclonal and monoclonal antibodies specific for the CP4 EPSPS arecommercially available from Strategic Diagnostics (Newark, Del.). CP4EPSPS can be detected from protein extracts of wheat 33391 plants, plantparts and seeds by immunological methods that include ELISA.

[0053] An ELISA procedure that uses 100 ng of monoclonal anti-CP4 EPSPSantibody diluted in 100 μl of 0.05 M carbonate-bicarbonate buffer pH 9.6is absorbed to the well of a microtiter plate overnight at 4° C. Thewell is washed with phosphate buffered saline 0.05% Tween-20, pH 7.4(PBS-T). The tissue is homogenized in phosphate buffered saline with amortar and pestle or other suitable tissue grinder. The homogenate isadded to the well of the microtiter plant and incubated for about 2hours at 37° C. The well is washed three times with PBS-T. In onemethod, a secondary antibody, a purified rabbit anti-CP4 EPSPS isdiluted to a sufficient level to provide specific binding to theCP4-EPSPS protein and incubated at 37° C. for about 1 hour. In a secondmethod, a secondary antibody, a goat anti-CP4 EPSPS is used. Abiotin-conjugated Mab anti-rabbit IgG or anti-goat IgG (Sigma Corp, StLouis Mo.) is added to the well (1:40,000 dilution in PBS) and incubatedat 37° C. for 30 minutes. The well is washed three times with PBS-T.NeutrAvidin conjugated Horse radish peroxidase is diluted 1:10,000 usingStabilZyme HRP-stabilizer (SurModics, Eden Prairie, Minn.) and incubatedat 37° C. for 15 minutes. The well is washed three times with PBS-T. TheTMB substrate (Kirkegaard and Perry, Gaithersburg, Md.) is added for 10minutes, then reaction quenched using 3 M phosphoric acid. The well isread with a microtiter plate reader at 450 nm using a referencewavelength of 650 nm. This method is an example of an ELISA suitable fordetection of CP4 EPSPS and is not intended to be the only ELISA methodthat can be used to detect CP4 EPSPS, those skilled in the art of ELISAwill know that variations to the method can be designed to provide adetection assay specific and sufficiently sensitive to detect CP4 EPSPSin a plant tissue extract.

[0054] ELISA of field grown forage of wheat event 33391 contain a meanlevel of 58.2±8.4 μg/g, with a range of 45.5 to 72.4 μg/g, CP4 EPSPSprotein on a fresh weight tissue (fwt) basis, while the non-transgeniccontrol forage had no detectable level of the CP4 EPSPS protein abovethe ELISA method's limit of detection at 0.9 μg/g fwt. Wheat event 33391grain tissues contain a mean level of 12.6±2.5 μg/g, with a range of 9.5to 17.6 μg/g, CP4 EPSPS protein on a fresh weight tissue (fwt) basis,while the non-transgenic control forage had no detectable level of theCP4 EPSPS protein above the ELISA method's limit of detection at 0.1μg/g fwt. ELISA or other immunological methods for detecting CP4 EPSPScan be used as a diagnostic test for wheat 33391, when wheat 33391progeny are the only USDA (United States Department of Agriculture)registered glyphosate tolerant wheat that expresses the CP4 EPSPSprotein.

[0055] A deposit of the Monsanto Company, wheat 33391 disclosed aboveand recited in the appended claims has been made under the BudapestTreaty with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110. The ATCC accession number isPTA-2347. The deposit will be maintained in the depository for a periodof 30 years, or 5 years after the last request, or for the effectivelife of the patent, whichever is longer, and will be replaced asnecessary during that period.

[0056] Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

[0057] All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1 12 1 32 DNA Oryza sativa 1 cgactcaaat acagatatgc atttccaaaa gc 32 2 30DNA Oryza sativa 2 gactatcccg actctcttct caagcatatg 30 3 27 DNAAgrobacterium tumefaciens 3 catgtaatgc atgacgttat ttatgag 27 4 30 DNAAgrobacterium tumefaciens 4 atcgcgcgcg gtgtcatcta tgttactaga 30 5 399DNA Artificial Sequence misc_feature (1)..(399) chimeric sequence ofwheat genome and transgene insert 5 acacacgtac ccccaaacga tcagaagaggagccaaaaac ctaattccac caccgcaagt 60 ttctgtatcc acgagatccc atcttggggcctgttccgga gctccgccgg acgagggccg 120 tcatcacgga tggcttctgc atcatcatagcctctccgat gaagtgtgag tagtttacct 180 cagaccttcg ggtccatagt tagtagctagatggcttctt ctctctcttt gaatctcaat 240 acaaagttct ccccctctct aattcggaaatctttatttc gacgtgtcta cattcacgtc 300 caaatggggg cttagatgag aaacttcacgatcgatgcgg ccgcgttaac aagcttactc 360 gaggtcattc atatgcttga gaagagagtcgggatagtc 399 6 431 DNA Artificial Sequence misc_feature (1)..(431)chimeric sequence of wheat genome and transgene insert 6 atcgcgcgcggtgtcatcta tgttactaga tcggggatat ccccagcttg atggggatca 60 gattgtcgtttcccgccttc agtttaaact atcagtgttt aaataattga tagaacctca 120 aataattatgacgatgtcca ggcactgatc aatacatagg catcacgtcg aagattagta 180 gatcgaagattagtagactg acgatgtcca ggcactgatc aatacatagg gatcggggat 240 aaccaaattactgttgggca attgatagaa cctcaaataa ttatgacgat gtccaggcac 300 tgatcaatacataggcatca cgtcgaagat tagtagaccg actccttcct gcatctacta 360 ctattactccacacatcgac agttatccag catacatcta gtgtattaag ttcatggaaa 420 aacggaaatg c431 7 24 DNA Triticum aestivum 7 cgatcagaag aggagccaaa aacc 24 8 26 DNAOryza sativa 8 cgactcaaat acagatatgc atttcc 26 9 24 DNA Triticumaestivum 9 cataatggga ggcatgcttc gctg 24 10 24 DNA Triticum aestivum 10ccggttctca ctgctatctg caac 24 11 22 DNA Artificial Sequence misc_feature(1)..(22) fully synthetic sequence 11 gtatatcgac tcactatagg gc 22 12 19DNA Artificial Sequence misc_feature (1)..(19) fully synthetic sequence12 actatagggc acgcgtggt 19

We claim:
 1. A method of improving glyphosate tolerance in a wheat plantcomprising: (1) constructing a DNA construct comprising a first and asecond expression cassette, wherein said first expression cassette inoperable linkage comprises (i) a rice actin 1 promoter; (ii) a riceactin 1 intron; (iii) a chloroplast transit peptide encoding DNAmolecule; (iv) a glyphosate tolerant EPSPS encoding DNA molecule; and(v) a transcriptional terminator DNA molecule; and said secondexpression cassette comprising in operable linkage (a) a CaMV 35Spromoter; (b) a Hsp70 intron; (c) a chloroplast transit peptide encodingDNA molecule; (d) a glyphosate tolerant EPSPS encoding DNA molecule; and(e) a transcriptional terminator DNA molecule; and (2) transforming awheat cell with said DNA construct; and (3) regenerating said wheat cellinto a wheat plant; and (4) treating said wheat plants with an effectivedose of glyphosate; and (5) selecting fertile wheat plants that arevegetative and reproductive tolerant to glyphosate.
 2. A fertileglyphosate tolerant wheat plant produced by the method of claim
 1. 3.The progeny seeds of the glyphosate tolerant wheat plant of claim
 2. 4.A DNA molecule isolated from the fertile glyphosate tolerant wheat plantof claim 2 comprising a nucleotide sequence identified as SEQ ID NO:5.5. A DNA molecule isolated from the fertile glyphosate tolerant wheatplant of claim 2 comprising a nucleotide sequence identified as SEQ IDNO:6.
 6. A pair of DNA molecules comprising: a first DNA molecule and asecond DNA molecule, wherein the first DNA molecule is of sufficientlength of contiguous nucleotides from the wheat genomic sequence of SEQID NO:5 or its complement to function as a DNA primer, and the secondDNA molecule is of sufficient length of contiguous nucleotides from theinsert sequence of SEQ ID NO:5 or its complement to function as a DNAprimer, and the pair of DNA molecules when used in a DNA amplificationmethod produce an amplicon diagnostic for DNA extracted from glyphosatetolerant wheat plant 33391 or progeny thereof.
 7. A pair of DNAmolecules comprising: a first DNA molecule and a second DNA molecule,wherein the first DNA molecule is of sufficient length of contiguousnucleotides from the wheat genomic sequence of SEQ ID NO:6 or itscomplement to function as a DNA primer, and the second DNA molecule isof sufficient length of contiguous nucleotides from the insert sequenceof SEQ ID NO:6 or its complement to function as a DNA primer, and thepair of DNA molecules when used in a DNA amplification method produce anamplicon diagnostic for DNA extracted from glyphosate tolerant wheatplant 33391 or progeny thereof.
 8. A method of detecting the presence ofa DNA molecule diagnostic for glyphosate tolerant wheat plant 33391 orprogeny thereof, the method comprising: (a) extracting a DNA sample fromsaid wheat plant 33391 or progeny seeds and plants or parts thereof; and(b) providing DNA primer molecules SEQ ID NO:7 and SEQ ID NO:8; and (c)providing DNA amplification reaction conditions; and (d) performing saidDNA amplification reaction, thereby producing a DNA amplicon molecule;and (e) detecting the DNA amplicon molecule.
 9. In the method of claim8, the DNA amplicon molecule comprising the DNA molecules of SEQ ID NO:7and SEQ ID NO:8.
 10. A method of detecting the presence of SEQ ID NO:5DNA molecule in a DNA sample, the method comprising: (a) extracting aDNA sample from a wheat plant; (b) contacting the DNA sample with a DNAmolecule that spans the junction region of wheat 33391 genomic DNA andinsert DNA of SEQ ID NO:5, wherein said DNA molecule is a DNA probe thathybridizes under stringent hybridization conditions with the DNAmolecule SEQ ID NO:5; and (c) subjecting the sample and probe tostringent hybridization conditions; and detecting hybridization of theprobe to the DNA.
 11. A method of detecting the presence of SEQ ID NO:6DNA molecule in a DNA sample, the method comprising: (a) extracting aDNA sample from a wheat plant; (b) contacting the DNA sample with a DNAmolecule that spans the junction region of wheat 33391 genomic DNA andinsert DNA of SEQ ID NO:6, wherein said DNA molecule is a DNA probe thathybridizes under stringent hybridization conditions with the DNAmolecule SEQ ID NO:6; and (c) subjecting the sample and probe tostringent hybridization conditions; and detecting hybridization of theprobe to the DNA.
 12. A method of breeding a glyphosate tolerant traitinto wheat plants comprising: a) crossing wheat 33391 glyphosatetolerant progeny with non glyphosate tolerant wheat plants; and b)producing progeny wheat plants of the cross; and a) extracting a DNAsample from progeny wheat plants; b) contacting the DNA sample with amarker nucleic acid molecule that hybridizes to SEQ ID NO:5 or SEQ IDNO:6 or complements thereof; and c) performing a marker assistedbreeding method for the glyphosate tolerant trait, wherein theglyphosate tolerant trait is genetically linked to a complement of themarker nucleic acid molecule.
 13. A DNA detection kit comprising: atleast one DNA molecule of sufficient length of contiguous nucleotideshomologous or complementary to SEQ ID NO:5 or SEQ ID NO:6 that functionsas a DNA primer or probe specific for wheat event 33391 and its progeny.14. A method of detecting wheat event 33391 and progeny thereofcomprising: (a) extracting a sample of wheat event 33391 for protein;and (b) assaying extracted protein by an immunological based methodcontaining antibodies specific for the CP4 EPSPS protein; and (c)detecting the reaction of the antibodies with the CP4 EPSPS protein. 15.A wheat seed designated 33391 and having ATCC Accession No. PTA-2347.16. A wheat plant or its parts produced by growing the seed of claim 15.17. A wheat plant of claim 16, wherein said wheat plant is tolerant toglyphosate.
 18. A wheat plant, or its parts, wherein at least oneancestor of said wheat plant is the wheat plant, or its parts, of claim17.
 19. A method of producing a wheat plant that tolerates applicationof glyphosate comprising: (a) sexually crossing a first wheat plantgrown from the wheat seed 33391 having ATCC Accession No. PTA-2347 thatconfers tolerance to application of glyphosate, and a second wheat plantthat lacks the tolerance to glyphosate, thereby producing a plurality offirst progeny plants; and (b) selecting a first progeny plant that istolerant to application of glyphosate; and (c) selfing said firstprogeny plant, thereby producing a plurality of second progeny plants;and (d) selecting from said second progeny plants a glyphosate tolerantplant second progeny plant.
 20. The method of claim 19 furthercomprising the steps of back-crossing the first progeny plant that isglyphosate tolerant or the glyphosate tolerant second progeny plant toany non-glyphosate tolerant wheat plant; and selecting progeny thereoffor tolerance to glyphosate.
 21. A method for selectively controllingweeds in a field containing a wheat crop having planted wheat 33391seeds or seeds of progeny thereof comprising the steps of: (a) plantingthe wheat 33391 seeds or progeny thereof that are glyphosate tolerant;and (b) applying to the wheat crop and weeds in the field a sufficientamount of glyphosate herbicide to control the weeds withoutsignificantly affecting the wheat crop.